Accumulating evidence implicates growth factors that bind to receptors with protein tyrosine kinase (PTK) activity in the pathogenesis of chronic renal failure. Recent evidence from this laboratory and others suggests that vasoactive peptides binding to G protein-coupled receptors constitute a novel group of growth factors. This class of growth factors (i.e., endothelin, angiotensin II) has also been implicated in diverse vascular disorders and in renal failure. But in contrast to growth factors that bind to receptor PTK, relatively little is known about the signals by which activated G protein-coupled receptors stimulate cell growth. The long-term goal of this project is to understand the signal transduction pathways mediating transcription and mitogenesis by growth factors that bind to G protein-coupled receptors. The focus is on endothelin (ET) peptides and cytosolic and nuclear effectors that lead to mitogenesis in glomerular mesangial cells. First, we will use pharmacological and molecular biological approaches to characterize the ET receptor subtype mediating mitogenic signaling. We will also test the hypothesis that protein kinase C (PKC) and PTK activity are necessary for stimulating the serum response cis-element and mitogenesis by ET-1. We have chosen to study the serum response element (SRE) because it is a dominant cis- element in the promoters of many immediate-early genes, whose expression are required for the G-o-G-l transition. PKC inhibition and depletion will be used to test whether PKC mediates the increase in SRE activity and mitogenesis by ET-1. SRE activity will be measured in transient transactivation assays with reporter constructs linked to the bacterial chloramphenicol acetyltransferase gene. We will also test the hypothesis that ET-1 stimulates a PTK activity, perhaps the nonreceptor PTK pp60c- src, and that this PTK activity is required for SRE activation and mitogenesis. The demonstration that ET-1 increases pp6Oc-src activity suggests that cross-talk between G protein-coupled receptors and non- receptor PTK contribute to mitogenic signaling. Activation of pp60c-src by ET-1 considerably enlarges on potential candidates for downstream effectors of ET receptors; potential effectors linked to pp60c-src include phosphatidyl inositol 3-kinase, ras GAP, and phospholipase C-gamma. The experiments outlined in this proposal will enable us to construct a molecular model of mitogenic signals evoked by ET-1. Predictions derived from this model might be useful in designing novel pharmacological interventions aimed at preventing the growth factor actions of ET-1. Because ET-1 has been implicated the vascular response to injury and in renal failure, the ability to block the mitogenic actions of ET might be relevant in clinical settings.